Efficacy of mRNA-1273 and Novavax ancestral or BA.1 spike booster vaccines against SARS-CoV-2 BA.5 infection in non-human primates

Omicron SARS-CoV-2 variants escape vaccine-induced neutralizing antibodies and cause nearly all current COVID-19 cases. Here, we compared the efficacy of three booster vaccines against Omicron BA.5 challenge in rhesus macaques: mRNA-1273, the Novavax ancestral spike protein vaccine (NVX-CoV2373), or Omicron BA.1 spike protein version (NVX-CoV2515). All three booster vaccines induced a strong BA.1 cross-reactive binding antibody and changed immunoglobulin G dominance from IgG1 to IgG4 in the serum. All three booster vaccines also induced strong and comparable neutralizing antibody responses against multiple variants of concern, including BA.5 and BQ.1.1, along with long-lived plasma cells in the bone marrow. The ratio of BA.1 to WA-1 spike-specific antibody-secreting cells in the blood was higher in NVX-CoV2515 animals compared to NVX-CoV2373 animals, suggesting a better recall of BA.1 specific memory B cells by the BA.1 spike-specific vaccine compared to the ancestral spike-specific vaccine. Further, all three booster vaccines induced low levels of spike-specific CD4 but not CD8 T cell responses in the blood. Following challenge with SARS-CoV-2 BA.5 variant, all three vaccines showed strong protection in the lungs and controlled virus replication in the nasopharynx. In addition, both Novavax vaccines blunted viral replication in nasopharynx at day 2. The protection against SARS-CoV-2 BA.5 infection in the upper respiratory airways correlated with binding, neutralizing, and ADNP activities of the serum antibody. These data have important implications for COVID-19 vaccine development, as vaccines that lower nasopharyngeal virus may help to reduce transmission.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of infections and deaths since 2019, with ongoing worldwide circulation still happening today (1,2).Its continued evolution has resulted in the emergence of a number of variants of concern (VOC), which has enhanced resistance to the immunity induced by current coronavirus disease 2019 (COVID-19) vaccines as well as greater replication fitness and transmissibility (3,4).In particular, the B.1.1.529(Omicron BA.1) VOC isolate contains 37 mutations in the spike protein, 15 of which are in the receptor binding domain (RBD) (5).Such mutations resulted in its rapid dominance, displacing prior variants to become responsible for >99% of infections in the United States in mid-January 2022 (1,2).Its sublineages of BA.2, BA.5, BA.2.75, and (more recently) BQ1.1 have even greater immune evasion and transmissibility (6), successively replacing the current Omicron variant to become dominant variants globally (1,2).Therefore, developing an effective COVID-19 vaccine that can restrict the emergence of SARS-CoV-2 VOCs and their transmissions remained a public health priority.
The waning of vaccine-induced immune responses is well reported in vaccinated individuals, with or without prior SARS-CoV-2 infection (7,8), and is a major concern for the current COVID-19 vaccines.Breakthrough infections after vaccination help drive ongoing SARS-CoV-2 transmission and the emergence of new VOCs (9).Current Food and Drug Administration (FDA)-approved COVID-19 vaccines provide incomplete protection against mild COVID-19 and transmission even shortly after booster vaccination, with waning protection against severe COVID-19 in the long term (1,2).Compared with the original primary series vaccination, booster doses provide a superior immune response against VOCs, including BA.1 Omicron and its sublineages (10), but these responses wane quickly (8,(11)(12)(13).With the multiple vaccines and immunization strategies available, along with waning antibody titers in circulation and mucosal compartments (1,2), it is critical to determine the best vaccine regimens to maximize durable immune responses against emerging VOCs.One strategy is to use a booster with a spike protein sequence matching circulating VOCs, but this has provided no advantage compared with boosting with the original wild-type sequence vaccine (14)(15)(16).
Durable mucosal immunity is a key component in protecting against SARS-CoV-2 and emerging VOCs as they infect the respiratory airways, primarily at the upper and lower respiratory mucosal surfaces (17,18).Heterologous subunit-based adjuvanted vaccines are known to induce robust and durable immune responses in the circulation and high immunoglobulin A (IgA) levels in the mucosae if given by a mucosal route (18)(19)(20).Use of the FDA-approved Novavax adjuvanted subunit-based protein vaccine NVX-CoV2373 as a booster after two-dose mRNA-1273 vaccination provides a good in vivo test case for mRNA-protein heterologous prime-boost.In addition, the induction of long-lived plasma cells (LLPCs) in bone marrow (BM) is vital in increasing the durability of serum antibody responses (21,22); therefore, it is crucial to develop vaccination approaches that maximize BM-LLPC production to protect against emerging SARS-CoV-2 VOCs.Nonhuman primate (NHP) studies are crucial in defining such vaccination strategies because NHPs are anatomically, physiologically, and behaviorally closer to humans.
Here, we conducted an NHP study to characterize the magnitude, breadth, and persistence of humoral and cellular immune responses induced by different booster vaccines in animals originally vaccinated with the two-dose mRNA-1273 primary series.Animals were boosted with either the homologous mRNA-1273 vaccine or adjuvanted protein-based vaccines from Novavax, NVX-CoV2373 (expressing WA-1 spike), and NVX-CoV2515 (expressing BA.1 spike).We characterized the magnitude, breadth, and durability of immune responses in the systemic and upper and lower airway mucosae collected before and after the second and third vaccination.We evaluated vaccine efficacy 3 months after the booster dose by challenging vaccinated and control NHPs with SARS-CoV-2 BA.5 Omicron VOC.The primary goals were (i) to compare the magnitude and breadth of antibody response induced by different booster vaccinations, (ii) to compare the longevity of antibody response induced by the booster with mRNA and adjuvanted NVX-CoV protein vaccines and how they influence protection against the SARS-CoV-2 BA.5 variant infection (most dominant VOC across the world at the time of the study) administered 3 months after the booster dose, and (iii) to determine whether there is benefit in using Omicron-specific spike during booster vaccination to provide protection against the Omicron variant.

All three booster vaccines induce a strong BA.1 crossreactive binding antibody with IgG4 dominance
Twenty-four Indian-origin male rhesus macaques (RMs), 3 to 5 years old, were divided into four groups (n = 6 per group) (Fig. 1A).Eighteen NHPs (groups 1 to 3) were administered the primary series of mRNA-1273 vaccine at weeks 0 and 4. At week 17, the group 1, 2, and 3 animals were boosted with mRNA-1273 (WA-1 matched spike, denoted in red), NVX-CoV2373 (WA-1 matched spike; denoted in blue), or NVX-CoV2515 (BA-1 matched spike; denoted in green), respectively.The NVX-CoV vaccines used in this study express full-length, prefusion-stabilized, spike (S) protein trimers and are formulated with a saponin-based adjuvant, Matrix-M.The fourth group of RMs was recruited at the time of challenge, did not receive any vaccination, and served as the control group (denoted in gray).All the immunizations were performed via the intramuscular route.To measure the protective efficacy, 3 months after the boost, all the RMs (vaccinated and unvaccinated) were challenged with the SARS-CoV-2 BA.5 VOC.Immunological analyses for control animals before challenge are not available because we recruited them at the time of challenge of vaccinated animals.
We measured binding antibodies against RBD and spike proteins derived from WA-1 and BA.1 VOCs using an enzymelinked immunosorbent assay (ELISA) at various times during vaccination.As expected and shown previously (23)(24)(25), the primary series of mRNA-1273 vaccinations induced strong RBD and spike binding antibodies at 2 weeks after the second dose (Wk6), and these responses contracted by about fivefold over the next 3 months (Fig. 1B).All three booster vaccines induced strong RBD and spike-specific binding antibodies.At week 19 (2 weeks after boost), the binding antibody response was boosted in all three groups.The mRNA-1273-vaccinated animals showed twofold higher WA-1 RBD binding antibodies compared with the NVX-CoV2515 vaccine (fig.S1A).Similarly, the mRNA-1273-vaccinated animals had 2.5-fold higher WA-1 S binding antibodies compared with both NVX-CoV vaccines (Fig. 1C).However, at week 27, the WA-1-specific RBD and S binding responses contracted by 8-fold (NVX-CoV groups) and 15-fold (mRNA group) and were comparable between the three groups (fig.S1, A and C).We next analyzed the cross-reactive binding to RBD and S proteins from BA.1 (figs. S1A and S2C).The binding to BA.1 RBD was 3.5-, 3-and 2.6-fold lower in the mRNA-1273, NVX-CoV2373, and NVX-CoV2515 groups, respectively, compared with binding to WA-1 RBD.Similarly, binding to BA.1 S was 2.4-, 1,8-, and 1.8-fold lower in the mRNA-1273, NVX-CoV2373, and NVX-CoV2515 groups, respectively, compared with binding to WA-1 S. This resulted in a relatively higher ratio of BA.1 to WA-1 S-specific antibody in both NVX-CoV groups, with a statistically significant difference between NVX-CoV2515 and mRNA-1273 groups (fig.S1B).
We next investigated the IgG subclass of RBD-binding antibodies at 2 weeks after the second and third (booster) doses.The primary mRNA-1273 vaccinations predominantly induced IgG1 (Fig. 1D), followed by IgG4 (Fig. 1E), with low levels of IgG2 (fig.S1C) and IgG3 (fig.S1D).However, the booster immunization predominantly induced an IgG4 response in all three groups.In addition, the NVX-CoV vaccines induced a 10-fold lower IgG1 response compared with the mRNA booster, with a statistically significant difference between the mRNA-1273 and NVX-CoV2515 groups.Consistent with a lower IgG1 response in the NVX-CoV groups, their antibodies had reduced binding to FcγRIIIA (Fig. 1F).Overall, these data demonstrate that the booster dose, regardless of vaccine, predominantly boosted the IgG4 response, and the NVX-CoV vaccines showed lower recall of IgG1 response (Fig. 1G).All three booster vaccines induced cross-reactive IgG1-and IgG4-binding antibody response against the spike and RBD proteins from other VOCs (Alpha, Beta, Delta, and Gamma and Omicron BA.1) with comparable response, except that the IgG1 response in the BA.1 vaccine was notably lower compared with the response induced by the mRNA-1273 vaccine (fig.S2).All three booster vaccines also showed similar targeting of the binding response to N-terminal domain, RBD, and S1 and S2 regions, which was predominantly directed to S1 and RBD regions (fig.S3).We further analyzed anti-RBD binding antibodies at week 19 (2 weeks after booster) in the nasal, pharyngeal, and bronchoalveolar lavage (BAL) fluids (n = 4 per group) using a binding antibody multiplex assay (BAMA) and ELISA (Fig. 1H and fig.S4).We found that all three vaccines induced comparable WA-1/2020 RBD IgG-specific activity (nanograms of antibody per microgram of total IgG) in mucosal secretions at the peak response (Fig. 1H).
Overall, these data demonstrate that (i) although the mRNA-1273 booster vaccine induced higher binding antibody responses than NVX-CoV protein boosters at the peak, the greater contraction in the mRNA-boosted group led to comparable titers in the memory phase (3 months after boost), (ii) booster vaccination induced an IgG4-dominant response, and (iii) BA.1 spike boost induced a relatively higher proportion of BA.1-specific response compared with WA-1-specific response.We next evaluated the neutralizing activity of sera.We performed a longitudinal analysis of WA-1/2020-specific 50% live-virus neutralization titers (ID 50 ) to understand the neutralizing antibody (NAb) responses induced after the second dose (week 6), third dose (week 19), and prechallenge (week 27) (Fig. 2 and fig.S5).Consistent with previous results (23)(24)(25), the two-dose mRNA-1273 vaccination induced a robust WA-1/2020-specific NAb titer at week 6 (2 weeks after the second dose) in all vaccinated NHPs (n = 18), with a geometric mean titer (GMT) of 6300.These responses contracted about 10-fold over 3 months to a titer of 680 (Fig. 2A).

All three booster vaccines induce low frequencies of spikespecific T cell response in the blood
We analyzed S (WA-1/2020)-specific interferon-γ-positive (IFNγ + ) CD4 and CD8 T cell responses in the blood (peripheral blood mononuclear cells) of vaccinated animals at weeks 2 (2 weeks after prime), 5 (1 week after the second dose), and 18 (1 week after the third dose) after prime immunization using an intracellular cytokine staining (ICS) assay (Fig. 3).Consistent with the previous studies (23)(24)(25), our analysis showed that mRNA-1273 elicits a low frequency of S-specific IFN-γ + CD4 T cell response at week 5 after the two-dose primary series of vaccination with a geometric mean of 0.07 (range, 0.01 to 0.24) (Fig. 3A).However, the magnitude of IFN-γ + CD4 T cell response at 1 week after the booster dose stayed low in all three groups (Fig. 3A).The group boosted with mRNA-1273 had significantly higher week 18 IFN-γ + responses compared with the two NVX-CoV groups (P = 0.011), indicative of a stronger T helper 1 (T H 1) response induced by the mRNA booster compared with the protein booster.S-specific interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) induction was also detected after the booster dose, but differences were not significantly different between groups (Fig. 3B).Thus, all vaccination strategies successfully induced T H 1 immune responses, with a significantly stronger IFN-γ + CD4 T cell response in the mRNAboosted group compared with the protein-boosted groups.The two primary series of mRNA vaccinations also induced low levels of IFN-γ + CD8 T cell responses in the blood, and the responses were marginally higher after the second immunization compared with the first immunization (Fig. 3C).However, after the booster dose, the responses were very low and mostly below our detection limit in all three groups (Fig. 3C).Similarly, the frequencies of TNFα-or IL-2-producing CD8 T cells were also very low after the booster immunization (Fig. 3D).Overall, these data indicated that booster vaccination with mRNA-1273, NVX-CoV-2373, and NVX-CoV-2515 after the primary series of mRNA-1273 vaccination induces low levels of S-specific CD4 but not CD8 T cell responses.

BA.1 vaccine induces greater proportion of BA.1-to WA-1specific plasmablast responses compared with WA-1 vaccine
Plasmablasts (PBs) or antibody-secreting cells (ASCs) in the blood after boost represent the magnitude, specificity, and diversity of memory B cells that are being recalled (33,34).In addition, the seeding of LLPC in BM is important to induce persisting serum antibody responses (21,35).We next investigated the effect of mRNA-1273, NVX-CoV2373, or NVX-CoV2515 boosting on the generation of spike-specific ASCs in the blood and LLPCs in BM.Analysis of WA-1/2020 spike-specific IgG + ASCs on days 0, 4, 7, and 28 after the booster vaccination showed a rapid increase on day 4 and a sharp decline on day 7 (Fig. 4A and fig.S6).Consistent with the serum antibody, frequencies of WA-1/2020 S-specific ASCs were about 3.6-fold higher in the mRNA-1273 group (geomean of 742) compared with the NVX-CoV2373 (geomean of 205) group at day 4 (Fig. 4B).Further, the ASCs induced by all three vaccines showed cross-reactivity to BA.1 spike, and the ratio of BA.1-to-WA-1 ASCs was twofold greater in the BA.1-specific vaccine compared with the WA-1-specific vaccine (Fig. 4C).The WA-1-specific ASCs at day 4 showed a positive correlation with WA-1 spike-specific IgG in serum at 2 weeks after the booster dose (week 19) (Fig. 4D).Analysis of RBD-specific ASCs also showed similar kinetics of ASC response (Fig. 4E), higher WA-1-specific response in the mRNA-1273 group (Fig. 4F), and a positive correlation with serum WA-1 RBD-specific IgG at week 19 (Fig. 4G).As expected, all three groups showed poor cross-reactive ASCs to BA.1 RBD (Fig. 4F).These data demonstrate that the mRNA-1273 vaccine provides a stronger boost of ASCs compared with NVX-CoV vaccine.They indicate that the NVX-CoV BA.1-specific vaccine recalled a greater proportion of BA.1-to WA-1-specific memory B cells compared with NVX-CoV WA-1-specific vaccine.They also suggest that a large portion of the BA.1 response was targeted outside of RBD.
Next, we evaluated LLPCs in BM on weeks 21 and 27 (4 and 10 weeks after the booster (Fig. 4, H to M).The frequency of LLPCs before the boost was below our detection limit; however, all three booster vaccines induced strong WA-1 spike-specific LLPCs at 4 weeks after the boost and showed a 3-to 6-fold decline at 10 weeks after boost (Fig. 4I).The responses were comparable between the three groups at both time points (Fig. 4J).Like PB responses, the LLPCs also showed some cross-reactivity to BA.1 spike (Fig. 4K) and the ratio of BA.1 to WA-1 S-specific LLPCs was greater in the BA.1-specific vaccine booster compared with the WA-1-specific vaccine booster (Fig. 4L).As expected, there was a strong correlation between S (WA-1/2020)-specific peak antibody at week 19 (2 weeks after booster) and week 21 LLPCs (4 weeks after booster) (Fig. 4M).Overall, these analyses showed that all booster vaccines induced strong LLPCs in BM and suggested that BA.1-specific vaccine induces a relatively higher proportion of BA.1-specific LLPCs.

All three booster vaccines protect against SARS-CoV-2 BA.5 infection in the lower airway
To determine the extent of protection provided by different booster vaccines, we challenged vaccinated and control NHPs at week 30 (3 months after the booster) with SARS-CoV-2 BA.5 via intranasal and intratracheal inoculation.BA.5 was the dominant VOC in mid-2022, with a spike sequence more closely related to BA.1 than to WA-1/2020 and with higher transmissibility and neutralization resistance compared with prior VOCs.Successive variant evolution has resulted in differential disease severity between different VOCs in animal models (36), and it is unknown whether BA.5 infects and causes pathogenesis in NHPs.
We measured nucleocapsid-specific subgenomic RNA (sgRNA N) in the nasopharynx (upper airway) and BAL (lower airway) on days 0, 2, 4, 7, and 10 (day of euthanasia) postinfection and compared the viral loads in the upper and lower airway between vaccinated and control animals (Fig. 5).We observed that the virus replication peaked at day 2 in both the BAL (Fig. 5B) and nasopharynges (Fig. 5E) of unvaccinated control NHPs, with the viral loads reaching a geometric mean copy number of 2.4 × 10 4 (Fig. 5A) and 1.2 × 10 8 (Fig. 5D) sgRNA N copies/ml, respectively.Five of six (5/ 6) in the lower airway and six of six (6/6) control NHPs in the upper airway showed virus replication.All animals had detectable viremia in the upper airway at all times tested until necropsy, along with 5/6 animals in the lower airway, indicating productive BA.5 VOC infection in NHPs.After the challenge, the mRNA-1273-, NVX-CoV2373-, and NVX-CoV2515-boosted animals showed markedly lower virus replication compared with controls in the lower (Fig. 5, A to C) and the upper (Fig. 5, D to F) airways, starting from day 2, with a significant difference on days 2 and 4 in the lower airways and until necropsy in the upper airways.Strikingly, in lower airways (BAL), all vaccinated animals showed profound viral control, with just one of six animals from the mRNA-1273 and NVX-CoV2515 groups having any detectable virus (Fig. 5B).None of the NVX-CoV2373-vaccinated animals had detectable viral loads in the lower airways (Fig. 5B).

NVX-CoV2373 and NVX-CoV2515 vaccines blunt viral replication in the upper airway
As has been observed with previous vaccines, the viral control was variable in the upper airways (23)(24)(25), yet three doses of vaccination lowered both the peak viremia and improved viral control at day 7 compared with the unvaccinated NHPs (Fig. 5, D to F).The heterologous vector-based booster vaccination with NVX-CoV2373 or NVX-CoV2515 resulted in significantly lower viral loads in the upper airways starting from day 2 and until necropsy, compared with unvaccinated animals (Fig. 5, D to F). Unexpectedly, a majority (four of six) of the mRNA-1273-boosted animals had variable but high viral loads (2.6 × 10 6 to 1 × 10 9 sgRNA N copies/ml) in the upper airways (Fig. 5E).Further, the NVX-CoV2373 booster provided superior protection in the upper airways, with virus detected only in three animals (Fig. 5E).
In addition, the area under the curve (AUC) of viral load in BAL and nasopharynx between days 0 to 10 was markedly lower in all the vaccinated animals compared with controls (Fig. 5, C and F).Similarly, enhanced protection from BA.5 infection was observed in the nasopharynges (upper respiratory airways) of NVX-CoV2373-and NVX-CoV2515-vaccinated animals compared with control animals (Fig. 5F).All the animals were euthanized on day 10 after the SARS-CoV-2 BA.5 challenge and evaluated for lung pathology and viral antigen as described previously (fig.S7, A to D, and table S1) (14,23,37).Four of the six control animals showed mild to moderate signs of inflammation, and two showed moderate to severe inflammation.Three of six controls also showed rare to occasional foci of viral antigen.In contrast, all of the NVX-CoV-vaccinated RMs, irrespective of the type of booster vaccine received, showed mild to moderate lung pathology, and 3 of 12 animals showed rare to occasional foci of viral antigen.However, three of six RMs from the mRNA-1273 booster group showed moderate to severe lung pathology and rare to occasional foci of viral antigen.In addition, body weights and respiratory rates remained stable after the challenge (fig.S8).Together, these data indicated that the booster vaccination with either mRNA-1273 or NVX-CoVs provides protection from virus replication in the airways.

Immune correlates for protection
Postchallenge at euthanasia, the binding (fig.S9A) and NAb (fig.S9B) responses were comparable between the three groups, and these responses were mostly below our detection limit in the unvaccinated controls.However, the control animals showed induction of spike-specific IFN-γ-, TNF-α-, and IL-2-producing CD4 T cells in the blood, BAL, lung, and hilar LNs, and these responses were A two-sided Mann-Whitney rank sum test was used to compare between control and respective vaccinated groups.ns, not significant; *P < 0.05; **P < 0.01.P values were not corrected for multiple comparisons because we did not make comparisons between two vaccine groups.The Spearman rank test was used for correlation analyses.
generally higher compared with vaccinated animals (fig.S10).The postchallenge antibody and T cell responses are consistent with profound viral control/protection in the vaccinated animals.
We performed correlations between various immune measurements postvaccination and viral control in the lower and upper airways (Fig. 5, G to K).We found some important associations with the sgRNA N loads in the nasal compartment and vaccineinduced antibody responses at weeks 19 (2 weeks post booster dose) and 27 (before challenge).The BA.5-specific neutralizing activity at week 19 and week 27 correlated inversely with day 7 viral loads (Fig. 5G).The binding IgG specific to BA-1 spike (Fig. 5H) and BA-1 RBD (Fig. 5I) at week 27, and BA.1-specific neutralizing activity at week 27 (Fig. 5J) and BA.4 Spike-specific ADNP activity at week 19 (Fig. 5K) also correlated inversely with day 7 viral loads.We found additional correlations between WA-1-specific binding activity, neutralization titer against WA-1 and BA.2.75, and day 7 viral loads (fig.S11).However, we did not find any significant associations with viral load in the lower airway because nearly all vaccinated animals showed viral loads below the detection level (fig.S12).These data suggested that multiple antibody functions, including binding, neutralizing, and ADNP activities, contributed to enhanced protection against SARS-CoV-2 BA.5 infection in the upper airway.
Overall, these results demonstrate that a booster with either the mRNA-1273, NVX-CoV2373, or NVX-CoV2515 vaccine protects from virus replication in the lower airway even after 3 months after vaccination, and a booster with both NVX-CoV2373 and NVX-CoV2515 vaccines provides superior viral control in the upper airway against BA.5 VOC infection in NHPs.

DISCUSSION
To the best of our knowledge, this is the first NHP study directly comparing an mRNA-1273 booster with an adjuvanted proteinbased vaccine booster in animals previously vaccinated with mRNA-1273.We designed this study to understand the magnitude, breadth, and durability of the antibody response induced by these two delivery platforms and their influence on the upper and lower airway protection against the heterologous BA.5 challenge administered 3 months after the booster.In addition, we designed the study to address whether a BA.1 spike-matched booster might perform better than the original strain against BA.5 challenge.Given that most human infections occur during periods of waning immunity, we elected to challenge animals 3 months after their final booster dose to better assess the maintenance of protection.This is in contrast to the majority of SARS-CoV-2 NHP studies conducted so far, which challenged animals during the peak of vaccine-induced antibody response at 4 weeks after the boost.Our results showed that all three booster vaccines enhance crossneutralizing activity against multiple VOCs, including BA.5 and BQ 1.1 (the most dominant VOCs at the current time), and provide strong protection in the lower airway.They also showed that Novavax vaccines blunt viral replication in the upper airway.Overall, these results highlighted the near-term (3 months) protective ability of mRNA-1273 and NVX-CoV2373 booster doses against BA.5 challenge.
A key question for COVID-19 vaccination strategy is to address whether there is a need to change the vaccine to match the spike in the circulating VOCs.Our results using the BA.1 variant-specific spike vaccine booster did not show significant benefit in enhancing the cross-neutralization against the BA variants and protection against BA.5.Our observations align well with recent reports where booster vaccination with mRNA-1273 or a VOC-matched vaccine both induce comparable NAb responses against the VOC and protect the lower airway against beta (B.1.351)or Omicron (BA.1:B.1.1.529)challenges (14,24).The BA.1 Omicron variant contains 32 spike protein mutations, most located in NAb epitopes (38,39).Because of this, we expect that memory B cells that are common to both WA-1 and BA.1 spikes will be preferentially boosted.This does not translate into higher NAb titer against BA.1 because the majority of serum antibody is derived from BM plasma cells that were predominantly specific to WA-1 spike.However, our PB analysis at day 4 after boost showed that the BA.1-specific booster recruited a relatively higher proportion of BA.1-specific memory B cells than WA-1-specific memory B cells.These results strongly suggest that a second booster dose with a BA.1-specific vaccine could potentially induce a higher magnitude of cross-reactive NAb responses against BA variants compared with an ancestral spike-specific boost.
One of the goals of our study was to compare the magnitude, breadth, and longevity of the immune response induced between the booster vaccines.Previous studies (14,15,24,40) showed that three doses of mRNA-1273 significantly improved the WA-1/2020specific neutralization titers compared with two doses, which we also observed here.In addition, all three booster vaccines enhanced the cross-reactive NAb titers against VOCs, including BA.1, BA.2.75, BA.2.75.2, BA.5, and BQ.1.1,with detectable neutralization titers 3 months after the booster dose and with no significant differences between booster vaccines.Although the mRNA-1273 booster showed a stronger NAb response against the ancestral WA-1 spike compared with the NVX vaccines, this was not true for NAb responses against other VOCs, highlighting some differences between the two platforms.There were no differences in the magnitudes of non-NAb responses such as ADCD, ADCC, and ADCP after two and three doses of vaccination, indicating that these responses were not as significantly boosted as the NAb response.This could be due to a change in the dominance of IgG subclass of the antibody from IgG1 to IgG4 after the boost.In general, we found that more vaccine doses appeared to be linked to the induction of higher spike-associated IgG4 antibody responses, irrespective of vaccine booster vaccine.Recent studies in humans also showed similar IgG4 dominance after booster immunizations with BNT162b2 and mRNA-1273 mRNA vaccinations (41)(42)(43).These results highlight similarities between human and NHP studies in modulating IgG class switching during vaccination.It was interesting to note that the booster vaccine type did not significantly alter the specificity of antibody response with respect to the major regions of spike, with the majority of the antibody response targeting the RBD region.Booster type did not alter binding to the RBD region in spite of the presence of many mutations within the Omicron RBD in NVX-CoV2515.Overall, the effect of the third dose was to improve NAb titers against VOCs and increase IgG4specific anti-spike antibodies, all without substantially changing domains being targeted on spike protein.Higher-resolution methods, such as electron microscopy-based polyclonal epitope mapping (EMPEM), may be needed to better characterize differences in antibody specificities between different vaccination regimens.
While NAb titers appear to be severely affected by mutations in the Omicron VOCs, studies have shown that T cell epitopes are more conserved, suggesting that T cell immunity plays a role in limiting severe disease in the absence of NAb epitopes (44)(45)(46).Consistently, our study showed that all three booster vaccinations induced similar CD4 T cell responses.We observed a bias toward T H 1 cells, which may be necessary for the control of SARS-CoV-2 VOCs.The IFN-γ + CD4 T cell responses were significantly higher in mRNA-1273 boosted animals than the combined NVX-CoV boosted animals (NVX-CoV2373 and NVX-CoV2515), suggestive of a stronger T H 1 response when boosting with mRNA.
One of the important findings of our study is that NVX vaccines blunt virus replication in the upper respiratory airway early after infection.All three vaccines showed a profound control of virus replication in the upper respiratory airway by 7 to 10 days, whereas the control animals showed high levels of persisting viral loads.Consistent with previous studies (23)(24)(25), the mRNA-1273 booster offered only limited protection early after infection (at days 2 and 4) in the upper airways.However, both NVX vaccines showed significant control of viral load in the upper airways starting from day 2 until necropsy.Nasopharyngeal viral loads correlate with the presence and quantity of infectious viruses (47); thus, vaccines that reduce viral loads early during infection are likely to help reduce transmission to other individuals.Our results highlight the need to study NVX vaccines' impact on viral load in the upper respiratory airway in a larger cohort of NHPs and humans.The mechanisms that contributed to enhanced viral control in the upper respiratory airways of NVX vaccinated animals are not completely clear.One possibility is that the NVX vaccines had higher levels of spike-specific IgG in the nasal secretions at the time of challenge.
In summary, our data demonstrate that a booster dose with either mRNA-1273 or NVX-CoV (2373 or 2515), after a primary series of mRNA-1273, elicits broadly cross-reactive and durable humoral immune responses and protects NHPs against SARS-CoV-2 BA.5 VOC infection.Our results suggest that the NVX-CoV2373 (WA-1 matched) booster provides greater protection in the airways, with superior viral control in the upper respiratory airways compared with controls.These data support the use of all currently available boosters for the prevention of infection and transmission of disease from circulating and emerging SARS-CoV-2 VOCs and suggest that adjuvanted protein boosters may be a preferred option to maximize protection.

Study design
We designed this study to evaluate the immunogenicity of mRNA-1273, NVX-CoV2373 (WA-1 spike matched), or NVX-CoV2515 (BA.1 spike matched) booster vaccination and their efficacy against SARS-CoV-2 BA.5 variant challenge in NHPs.To mimic a significant fraction of SARS-CoV-2-vaccinated individuals in the US, we vaccinated animals initially with two doses of mRNA-1273 to establish a primary series of vaccination before the booster dose was administered.We challenged animals with SARS-CoV-2 BA.5 VOC because it was the dominant VOC across the world at the time of challenge.We characterized the magnitude and breadth of cellular and humoral immune responses after vaccination and challenge to define immune correlates for protection against heterologous SARS-CoV-2 BA.5 infection.We used six animals per group on the basis of our previous experience in detecting immune responses and assessing protective efficacy in vaccine studies in macaques.We collected various samples, including blood, mucosal swabs (nasal and throat), BAL fluid, and BM multiple times postvaccination and challenge.In general, these included collections on the day of and 1 to 2 weeks after each vaccination and at euthanasia (day 10 postchallenge).After the challenge, we collected samples from the nasopharynx and BAL every 2 to 3 days until euthanasia to measure the viral loads in the upper and lower respiratory tract, respectively.We collected blood, BAL, and lung tissue at necropsy to evaluate postchallenge cellular and humoral immune responses.In addition, we assessed lung pathology and the SARS-CoV-2 antigens at euthanasia using histology on the day and necropsy.

Experimental design RM model and immunizations
Twenty-four Indian-origin male RMs (Macaca mulatta), 3 to 5 years old, were evenly divided into four groups of six animals each, matched on age and weight.Groups 1 to 3 were administered the primary series of two doses of 100 μg of mRNA-1273 (WA-1matched spike) at weeks 0 (prime) and 4 (boost).At week 17, groups 1, 2, and 3 were boosted with 50 μg of mRNA-1273 (WA-1 matched spike), 5 μg NVX-CoV2373 (WA-1 matched spike), and 5 μg NVX-CoV2515 (BA-1 matched spike), respectively.Groups 2 and 3 also received 50 μg Matrix-M adjuvant as part of their protein vaccines.The fourth group of RMs was recruited at the time of challenge, received no vaccine, and served as the control group.NVX-CoV2373 and NVX-CoV2515 were co-formulated with Matrix-M adjuvant, and single-dose vials were shipped at 2 to 8°C.All immunizations were performed via the intramuscular route, and a conventional 25gauge needle (0.5-or 1-ml volume) was used to deliver the vaccines.

SARS-CoV-2 BA.5 challenge in RMs
The twenty-four RMs (vaccinated and unvaccinated) were reorganized into five viral challenge cohorts.Each cohort contained a mix of unvaccinated and vaccinated animals, except cohorts 1 (n = 3, all controls) and 2 (n = 3, all vaccinated), which contained three RMs per group.Cohorts 3, 4, and 5 had six RMs per group, which included one control and five vaccinated animals.All animals were housed at the Emory National Primate Research Center (ENPRC) of Emory University under BSL2 conditions during the vaccination phase and were moved to a BSL-3 suite at least 1 week before the challenge for acclimatization.The time interval between the final vaccination and challenge was 13 weeks, 5 days for all animals except for the three vaccinated animals in challenge cohort 2, where the interval was between 12 to 14 weeks.
At week 30, 13 weeks after the final immunization, the macaques were challenged with a total of 6 × 10 5 PFUs of the SARS-CoV-2 BA.5 variant (Omicron BA.5 VOC, titered on Vero-TMPRSS2 cells).The virus was administered as 2 ml by the intratracheal route and 1 ml by the intranasal route (0.5 ml in each nostril).Nasopharyngeal swabs and BAL samples were collected, stored immediately in an RNA/DNA shield, and processed for viral RNA extraction.After the viral challenge on day 0, nasopharyngeal swabs and BAL fluid were collected in an RNA/DNA shield on days 2, 4, 7, and 10, and their viral loads were measured.On day 10, after the SARS-CoV-2 challenge, all vaccinated and nonvaccinated macaques were euthanized.Necropsy samples (lung tissues) were collected and stained with hematoxylin and eosin.
Immune complexes were then washed in PBS with 0.1% BSA and 0.02% Tween 20.Murine secondary antibodies for each antibody isotype or subclass were used to detect antigen-specific antibody titer and probed with a phycoerythrin (PE)-conjugated tertiary anti-mouse antibody (Invitrogen).For FcγRs, biotinylated FcγRs were labeled with streptavidin-PE before addition to immune complexes.Fluorescence was measured with an iQue (Intellicyt) and analyzed using Forecyt software.Data are reported as MFI.ADCP, ADNP, and ADCD assays for monkey sera ADCP, ADNP, and ADCD were measured as previously described in (58)(59)(60).Neutravidin beads were coupled to biotinylated SARS-CoV-2 antigens.Antigen-coupled beads were then incubated with appropriately diluted plasma (ADCP, 1:200; ADNP, 1:50; ADCD, 1:10) for 2 hours at 37°C to form immune complexes.For ADCP, 2.5 × 10 4 THP-1s cells were added and incubated for 16 hours at 37°C . For ADNP, leukocytes were isolated from fresh peripheral whole blood by lysing erythrocytes using ammonium-chloride potassium lysis.Leukocytes were added to immune complexes at 5 × 10 4 cells per well and incubated for 1 hour at 37°C.Neutrophils were detected using anti-human CD66b Pacific-Blue.For ADCD, lyophilized guinea pig complement was resuspended, diluted in gelatin veronal buffer with calcium and magnesium (GVB++, Boston Bio-Products), and added to immune complexes.The deposition of C3 was detected using an anti-C3 fluorescein isothiocyanate antibody.
All functional assays were acquired with an iQue (Intellicyt) and analyzed using Forecyt software.For ADCP, events were gated on singlets and fluorescent cells.For ADNP, bead-positive neutrophils were defined as CD66b-positive fluorescent cells.For both ADCP and ADNP, a phagocytic score was expressed as (percentage of bead-positive cells) × (MFI of bead-positive cells) divided by 10,000.For ADCD, data were reported as median fluorescence of C3 deposition (MFI).

ICS assay
Functional responses of SARS-CoV-2 S1-and S2-specific CD8 + and CD4 + T cells in vaccinated animals were measured using peptide pools and an ICS assay.Please refer to the Supplementary Materials for detailed methods.

ELISpot assay
The enzyme-linked immunosorbent spot (ELISpot) assays were performed as previously described (61) with few modifications.Please refer to the Supplementary Materials for detailed methods.
Viral RNA extraction and quantification SARS-CoV-2 subgenomic RNA was quantified in nasopharyngeal swabs, throat swabs, and BAL.Please refer to the Supplementary Materials for detailed methods.

Histopathology and immunohistochemistry
Histopathology and detection of SARS-CoV-2 virus antigen were performed as previously described (14,23,37).Briefly, lung tissue sections were processed and stained with hematoxylin and eosin for pathological analysis and with a rabbit polyclonal anti-SARS-CoV-2 antinucleocapsid antibody in immunohistochemistry staining for the presence of virus antigen.The polyclonal antibody (GeneTex, GTX135357) was used at a dilution of 1:2000.The tissue sections used for gross histology examination include the left cranial lobe (Lc), right middle lobe (Rmid), and right caudal lobe (Rc).The extent and severity of alveolar inflammation were characterized by the following criteria: the number of lung lobes affected, type 2 pneumocyte hyperplasia, alveolar septal thickening, fibrosis, perivascular cuffing, peribronchiolar hyperplasia, inflammatory infiltrates, and hyaline membrane formation.Each lung lobe was assessed individually for animals with multiple affected lung lobes, and then the scores were by presence and absence of signs for inflammation and virus antigen.Tissue sections were analyzed by a blinded board-certified veterinary pathologist using an Olympus BX43 light microscope.Photomicrographs were taken on an Olympus DP27 camera.

Quantification and statistical analysis
Throughout the manuscript, we compared mRNA-1273 and NVX-CoV2373 groups to determine differences between the two platforms and NVX-CoV2373 and NVX-CoV2515 groups to determine differences between the two spike proteins.Accordingly, we used a threshold P value of less than 0.025 as being significant to correct for multiple comparisons.The difference between any two groups at a time point was measured using a two-tailed nonparametric Mann-Whitney rank-sum test.Comparisons between different time points within a group used paired parametric t tests.A P value less than 0.05 was considered significant for comparisons between different time points within a group.The sample n is listed in corresponding figure legends.The correlation analysis was performed using the Spearman rank test.GraphPad Prism version 8.4.3 (GraphPad Software) was used for data analysis and statistics.

Supplementary Materials
This PDF file includes: Materials and Methods Figs.S1 to S12 Table S1 Other Supplementary Material for this manuscript includes the following: Data file S1 MDAR Reproducibility Checklist View/request a protocol for this paper from Bio-protocol.

Fig. 1 .
Fig. 1.Binding antibody responses in the blood and mucosal secretions.(A) Schematic of the nonhuman primate study.Eighteen NHPs were vaccinated with mRNA-1273 at weeks 0 and 4. At week 17, animals were divided into three groups (n = 6 per group).Groups 1, 2, and 3 received mRNA-1273, NVX-CoV2373, or NVX-CoV2515, respectively.An additional six animals were recruited at the time of challenge (group 4), did not receive vaccination, and served as the control group.At week 30, all the animals were challenged with the SARS-CoV-2 BA.5 variant via intranasal and intratracheal routes and euthanized at day 10 after challenge.(B) RBD (WA-1)-specific IgG antibodies in the serum.Data are means ± SEM for each group.(C) WA-1 and BA.1 spike-specific binding antibody titer at weeks 19 and 27 in serum.(D to F) Spike-specific IgG1 (D), IgG4 (E), and FCγRIIIA binding (F) responses at week 19 in serum.(G) Distribution of IgG subclasses at weeks 6 and 19.(H) RBD (WA-1)-specific IgG antibody response in mucosal (nasal, throat, and lung lavage) secretions at week 19.Each dot indicates one monkey (n = 6 per group except for BAL fluids where data are available only for n = 4 per group).Data represent one independent experiment.Each sample was analyzed in duplicate.Whiskers on dot plots show the maximum and minimum values.Horizontal dotted lines indicate assay limits of detection (C) or geometric mean value at week 0 of the study (D to F).A two-sided Mann-Whitney rank sum test was used to compare between groups.*P < 0.025.

Fig. 2 .
Fig. 2. Homologous and cross-reactive functional antibody responses in the blood.(A) WA-1-specific live-virus NAb titer in serum.Data are geometric means with error.(B) WA-1/2020-and BA.1-specific NAb titer for individual animals at weeks 17, 19, and 27.The numbers on the top and bottom on the graph represent the GMT at each time point.The numbers with x values indicate ratios of week 19 and 27 Nab titer relative to week 17 titer.(C) BA.1-to-WA-1 NAb titer ratios at weeks 19 and 27.(D) WA-1-and cross-reactive BA.1, BA.2.75, BA.5, BA.2.75.2, and BQ.1.1VOC-specific NAb titer at week 19.The numbers on the top and bottom on the graph represent the GMT.The numbers with bold font indicate fold change compared with WA-1.(E to G) WA-1 S-specific non-NAb effector functions: ADNP (E), ADCP (F), and ADCD (G) at weeks 6 and 19 and cross-reactive ADCD (H) activity at week 19.Each dot indicates one monkey (n = 6 per group).Data represent one independent experiment.Each sample was analyzed in duplicate.Whiskers on dot plots show maximum and minimum values.Horizontal dotted lines indicate assay limits of detection (B and D) or geometric mean value at week 0 of the study (E and H).

Fig. 4 .
Fig. 4. Antigen-specific ASCs in blood and bone marrow plasma cells.(A) WA-1 spike-specific ASCs measured by ELISpot on days 0, 4, 7, and 28 after the booster dose for each group.(B) WA-1 and BA.1 spike-specific ASCs for individual animals at day 4. (C) Ratios of BA.1 to WA-1 spike-specific ASCs at day 4. (D) Correlation between spike-specific binding antibody and ASCs.(E) WA-1 RBD-specific ASCs on days 0, 4, 7, and 28 after the booster dose for each group.(F) WA-1 and BA.1 RBD-specific ASCs for individual animals at day 4. (G) Correlation between RBD-specific binding antibody and ASCs.(H) Representative ELISpot images of bone marrow plasma cells (BMPCs).(I) S (WA-1)-specific BMPC responses at weeks 15, 21, and 27.(J to L) WA-1 and BA.1 S (J)-specific BMPCs and their ratios (K) at weeks 21 and 27.(L) BA.1 to WA-1 S-specific BMPC ratios at weeks 21 and 27.(M) Correlation between S (WA-1)-specific peak antibody (week 19) and BMPCs (week 21).Each dot indicates one monkey (n = 6 per group).Data represent one independent experiment.Each sample was analyzed in duplicate.Whiskers on dot plots show the maximum and minimum values.Dotted lines indicate assay limits of detection.Data in (A), (E), and (I) show means ± SEM.A two-sided Mann-Whitney rank sum test was used to compare between groups.*P < 0.025.The Spearman rank test was used for correlation analyses.

Fig. 5 .
Fig. 5. Efficacy of booster vaccines against BA.5 infection in respiratory airways.Subgenomic nucleocapsid RNA (sgRNA N) of SARS-CoV-2 BA.5 VOC viral loads (sgRNA N copies/ml) in BAL (A to C) and nasal swabs (D to F) after challenge.Viral loads are shown as geomean for each group (A and D), for individual animals (B and E), and as the area under the curve (AUC) (C and F).The AUC was calculated using viral load from days 0 to 10.Each dot indicates one monkey [n = 6 per group except on day 10 postinfection (necropsy) nasal swabs, where data are available for five animals in the mRNA-1273 (n = 5 per group) and four animals in NVX-CoV2373 (n = 4 per group) groups].Data represent one independent experiment.Each sample was analyzed in duplicate.Whiskers on dot plots show the maximum and minimum values.Dotted lines in (A), (B), (D), and (E) indicate assay limits of detection.(G to K) Correlation between day 7 viral load in nasal swabs and week 19 and week 27 BA.5 NAb titer (G), S (BA.1)-specific IgG (H), RBD (BA.1)-specificIgG (I), week 27 BA.1 NAb titers (J), and week 19 BA.4 spike--specific ADNP activity (K).A two-sided Mann-Whitney rank sum test was used to compare between control and respective vaccinated groups.ns, not significant; *P < 0.05; **P < 0.01.P values were not corrected for multiple comparisons because we did not make comparisons between two vaccine groups.The Spearman rank test was used for correlation analyses.